View
218
Download
2
Category
Tags:
Preview:
Citation preview
Sabine Franke, PhDCHU Liège, Centre for Human Genetics
20/04/12 ULG
Acquired alterations of
IGH and TCR loci in lymphoproliferative
disorders
Interuniversity course - HUMAN GENETCIS
Overview
what are lymphoproliferative disorders?
what are IGH and TCR?
what are alterations and how to detect them?
Lymphoproliferative disorders (LPDs)
LPDs refer to several conditions in which lymphocytes are produced in excessive quantities.
Chronic lymphocytic leukemia Acute lymphoblastic leukemia Hairy cell leukemia lymphomas Multiple myeloma Waldenstrom’s macroglobulinemia Wiskott-Aldrich syndrome Post-transplant lymphoproliferative disorder Autoimmune lymphoproliferative syndrome (ALPS) ‘Lymphoid interstitial pneumonia’
Lymphoid malignancy B lineage T lineage NK lineage
Acute lymphoblastic leukemia– children 82 – 86% 14 – 18% < 1%– adults 75 – 80% 20 – 25% < 1%
Chronic lymphocytic leukemias 95 – 97% 3 – 5% 1 – 2%
Non-Hodgkin lymphomas– nodal NHL 95 – 97% 3 – 5% < 2%– extranodal NHL 90 – 95% 5 – 10% < 2%– cutaneous NHL 30 – 40% 60 – 70% < 2%
Multiple myeloma 100% 0% 0%
B, T, and NK lineage of lymphoid malignancies
The ability to produce billions of different antibodies in humans results from the production of variable regions of light and heavy antibody genes by DNA rearrangement.
The five major classes of heavy chain are IgM, IgG, IgA, IgD, and IgE.
http://www.biology.arizona.edu
Identify lymphocyte populations derived from one single cell using the unique V-J gene rearrangements present within these antigen receptor loci.
These gene rearrangements generate products that are unique in length and sequence in each cell.
(junctional region)
stepwise rearrangement of V, D, and J gene segments
Genes encoding antigen receptors are unique:-high diversity -developing lymphocytes through V(D)J rearrangement.
The production of variable regions of light and heavy antibody genes by DNA rearrangement.
T-cell receptor gene rearrangement
Receptor for antigen on the majority of mature T-cells consists of two polypeptides alpha and beta that are linked by disulphide bonds and are associated with CD3
A small population of mature T-cells express a different TCR heterodimer in association with CD3. This is composed of two polypeptides designated gamma and delta
The variable domain of both the TCR α-chain and β-chain have three hypervariable or complementary determining regions (CDRs)
So how much variation is possible through recombining gene fragments?
Over 15,000,000 combinations of variable, diversity and joining
gene segments are possible.
Imprecise recombination and mutation increase the variability
into billions of possible combinations.
Estimated diversity of human Ig and TCR molecules
IgH Igα Igג TCR α β γ δ molecules molecules
Number gene segmentsV gene segments ~44 ~43 ~38 ~46 ~47 ~6 ~6D gene segments 27 2 3J gene segments 6 5 4 53 13 5 4
Combination diversity >2x10 6 2x10 6 <5000
Junctional diversity ++ + + + ++ ++ ++++
Total diversity >10 12 >10 12 >10 12
Clinically relevant testing
• Reactive versus malignant
• B-cell versus T-cell malignancy
• New lymphoma versus recurrence
• Assessment of remission and relapse
• Clinically relevant: bone marrow involvement (relation with prognosis)
• Evaluation of treatment effectiveness detection of minimal residual disease - treatment
PCRDiscrimination between monoclonal and polyclonal Ig/TCR gene PCR products
GeneScanning analysisFast, accurate, sensitivenon-quantitative monitoring of clonal proliferationsNeed sequence equipment
Heteroduplex analysisSensitivity ~5-10%
available to most laboratories
Design of novel primer sets for detection of Ig/TCR rearrangements
BIOMED-2 study (multiplex PCR)
Ig genes: IGH: VH-JH and DH-JH IGK: V-J and Kde rearrangements IGL: V-J
TCR genes: TCRB: V-J and D-J TCRG: V-J TCRD: V-J, D-D, D-J, and V-D
BIOMED-2 clonality strategy
Suspected B-cell lymphoma
Suspected lymphoma of unknown origin
SuspectedT-cell lymphoma
IGH V(D)J FR1, FR2, FR3IGH DJ(A)IGK-VJ and DE
TCRGVJ (A and B)TCRB V(D)J (A and B)
TCRB DJ
polyclonallym phocytes
m onoclonalleukem ia
IG H tube C : V -FR 3H J H
IG H tube C : V -FR 3H J H
BIO M ED -2 C oncerted Action BM H 4-C T98-3936: PC R-based clonality stud ies
PCR GeneScan analysis
region
region
IG H tube B V -FR 2–JH H
Mw
M
PB
-MN
C
ton
s il
ES
-6G
BN
-10
FR
-5
200 -
300 -
400 -
500 -600 -
700 -
800 -
bp 50100
0
150
100 400300200
100200
0
300
100 400300200
12502500
0
3750
100 400300200
15003000
0
4500
100 400300200
15003000
0
4500
100 400300200
250-295 nt
D H JH
J prim erHV -FR 2 prim ersH
V H
BIO M ED -2 report: Leukem ia 2003; 17: 2257-2317
tonsil
ES-6
G BN -10
FR -5
PB-M N C
Example BIOMED-2 multiplex IGHVH-FR2–JH
Use of controls
From the patient to the analysis
Selection of material protocol Check DNA quality Clonality analysis
Case 1
Female 63 years Lymphoma in dec. 2004 FR1 polyclonal, FR3 monoclonal Feb. 2009 biopsy Relapse?
Results
Controls okB-cell targets: IGH(VDJ) FR3 clonal
Molecular conclusion:Clonal rearrangement of the IGH gene was detected in this specimen.This gene rearrangement profile is identical to the one detected in the biopsie of 12/2004 and confirms the relapse of the disease.
Results
Controls okB-cell targets: IGH(VDJ) FR1 monoclonal
FR2 monoclonal
Molecular conclusion:Clonal rearrangements of the IGH gene were detected in this specimen.This gene rearrangement profile fits to the presense of a monoclonal B-cell population/B-NHL.
J 1.1J 1.2J 1.3J 1.4J 1.5J 1.6J 2.2J 2.6J 2.7
G TG G TC TAAG TG TC AAC ATC C ATTCC TG G TC C AATTG G C AAC ATC C ATTCTTC AAC C G AG TG AC AAC ATC C ATTCC TTG G G TC G AG AG AC AG AAC C C ATACC TG AG C TG AG AG G TAG G ATC C ATTCG TC C G AG TG AC AC TG TC C ATACTC C G AC TG G C ATG AC C C ATTCTC C G AC TG G C AC G AC C C G C TCG TC C G AG TG C C AATG TC C ATTC
(+53)(+53)(+55)(+56)(+55)(+58)(+56)(+58)(+52)
3 ' 5 '
D J
V fam ily prim ers J prim ers
TC R B tubes A and BV 2V 4V 5/1V 6a/11V 6b/25V 6cV 7V 8aV 9V 10V 11V 12a/3/13a/15V 13bV 13c/12b/14V 16V 17V 18V 19V 20V 21V 22V 23/8bV 24
(-204)(-201)(-197)(-201)(-201)(-201)(-198)(-201)(-198)(-201)(-198)(-198)(-198)(-198)(-201)(-198)(-201)(-201)(-193, )(-201)(-201)(-201)(-197)
inv
AAC TATG TTTTG G TATC G TC A C AC G ATG TTC TG G TAC C G TC AG C A C AG TG TG TC C TG G TAC C AAC AG AAC C C TTTATTG G TAC C G A C A ATC C C TTTTTTG G TAC C AAC AG AAC C C TTTATTG G TATC AA C AG C G C TATG TATTG G TAC AAG C AC TC C C G TTTTC TG G TAC AG AC AG AC C G C TATG TATTG G TATAAAC AG TTATG TTTAC TG G TATC G TAAG AAG C C AAAATG TAC TG G TATC AAC AA ATAC ATG TAC TG G TATC G AC AAG AC G G C C ATG TAC TG G TATAG AC AAG G TATATG TC C TG G TATC G AC AAG A TAAC C TTTATTG G TATC G AC G TG T G G C C ATG TAC TG G TAC C G AC A TC ATG TTTAC TG G TATC G G C AG TTATG TTTATTG G TATC AAC AG AATC A C AAC C TATAC TG G TAC C G AC A TAC C C TTTAC TG G TAC C G G C AG ATAC TTC TATTG G TAC AG AC AAATC T C AC G G TC TAC TG G TAC C AG C A C G TC ATG TAC TG G TAC C AG C A
5' 3 ’
V
TC R B tubes A and C : J A prim ers
J 2.1J 2.3J 2.4J 2.5
AG TG G C AC G ATC C ATTC TTC CAC TG TC AC G AG C C ATTC G C C CAG AG TC AC G AC C C ATTC G AC CC AC G AG C C AC AC G C G C
(+59)(+58)(+59)(+57)
3 ' 5 '
TC R B tubes B and C : J B prim ers
D J
D 1 prim er J prim ersD 2 prim er
(-252)
TC R B tube CD 1 G C C AAAC AG C C TTAC AAAG AC
5' 3 'D 2 (-137) TTTC C AAG C C C C AC AC AG TC
5' 3 '
BIO M ED -2 report: Leukem ia 2003; 17: 2257-2317
Analysis of TCRB gene rearrangements
D J
V fam ily prim ers J B prim ers
V
BIO M ED -2 report: Leukem ia 2003; 17: 2257-2317
Mw
M
PB
-MN
C
thym
us
Cel
l lin
e P
EE
R
ES
-9
ES
-6
TC R B tube B V -J
200 -
400 -
bp
800 -
250500
200 400300
0
750
450900
200 400300
0
1350
400800
200 400300
0
120016002000
7001400
200 400300
0
2100
ho
240-285 n t
he
ss
PB-M N C
cell line PEER
ES-6
ES-9
BIOMED-2 multiplex TCRB tube B
Use of clonality analysis
1. Making the diagnosisNormal reactive malignant
2. Involvement (staging)
3. Assessment of remission and relapseNormal reactive malignant
4. Evaluation of treatment effectiveness- detection of minimal residual disease (MRD)
MRD-based risk-group stratification (treatment reduction or treatment intensification)
Southern blot
Large region Large quantity of high molecular weight DNA Less sensitive Labor intensive
FISH – Fluorescence in situ hybridization
Relative large region Translocation partner has not te be known On metaphases and nuclei
breakpointreg ion
G ene A
no translocation translocation
Advantages:- detection of aberrations is independent of partner genes- m inim isation of fa lse positiv ity- identification of partner gene (or chrom osom e region), if m etaphases are present
Split-signal FISH
IG H gene com plex (#14q32.3)
1
V 3H V 66H D H JHV 2HV 1H
2 3 4 1 32 456
s s s sC C C 3 C 1 C 1
s s C 2 C 4 C 2s C s
IG K gene com plex (#2p11.2)V 1 V 2 V 3 V 76
1 32 4 5
J C Kde
IG L gene com plex (#22q11.2)JV 56V 3V 2V 1 J J C 3C 2C 1 J J J 654 J C 7
27
iE
3’E
iE 3’Eintr
on
RS
S
E
IG H -U(612 kb)
IG H -D(460 kb)
IG K -U IG K -D
IG L -U(309 kb)
IG L -D(343 kb)
IG L -U(520 kb)
Split-signal FISH for human Ig genes
TC R A TC R D and gene com plex (#14q11.2)
TC R B gene com plex (#7q34)
TC R G gene com plex (#7p14)
V 1 V 2 V 3 V 4 V 5 V n V 1 Rec
V 2 D J C
J J C
V 1 V 2 V 3 V 4 V 5 V n D 1 J 1 C 1 D 2 J 2 C 2
1 2 3 1 24 3
1 2 3 4 5 6 71 2 3 4 5 6
V 3
V 2 V 3 V 4 V 5 V 7 V 10 V 11V 8 V 9 J 1 C 1 J 2 C 2
1 2 3 31
functional V : 46 functional J : 53
functional V : 47
functional V : 6
TCR A/D -U TCR A/D -D
TCR B -U TCR B -D~20 kb
TCR G -U TCR G -D
Split-signal FISH for human TCR genes
Chromosome Genes Effect Occurrence Prognosis aberration involvedt(1;19)(q23;p13) E2A-PBX1 fusion 30% pre-B-ALL intermediate
t(4;11)(q21;q23) MLL-AF4 fusion 40% infant ALL poor
t(9;22)(q34;q11) BCR-ABL fusion 35% adult ALL poor
t(12;21)(p13;q22) TEL-AML1 fusion 25% childhood ALL good
del(1p32) SIL-TAL1 TAL1 15% childhood T-ALL intermediate
t(8;14)(q24;q32) IGH-MYC MYC 90% Burkitt’s lymphoma good
t(11;14)(q13;q32) BCL1-IGH Cyclin D1 >90% MCL poor
t(14;18)(q32;q21) BCL2-IGH BCL2 >90% FCL intermediate
t(2;5)(p23;q35) NPM-ALK fusion 50% ALCL intermediate
Prognostic value of chromosomal aberrations
FISH
• Excellent diagnostic tool for detection of well-defined chromosome aberrations
• Can also be used on tissue sections
• Requires limited handlingsusage of directly labeled probes
• Split-signal FISH has several major advantages– detection of aberrations, independent of partner gene– minimization of false-positive results – identification of partner gene or affected chromosome region, if metaphases are available
Summary
Gene rearrangements of the antigen receptor
genes occur
during the lymphoid proliferation These gene rearrangements generate products
that are
unique in length and sequence in each cell. Unique length allows by PCR discrimination of
monoclonality and polyclonality Standardized protocol (BIOMED-2)
Recommended